Chondroitin AC lyase

 

Glycosaminoglycans (GAGs) are highly negatively charged polysaccharides, formed from disaccharide repeating units. For a number of bacterial species, including Flavobacterium heparinum synthesise GAG lyases, these enzymes are used to degrade and utilise glycosaminoglycans as a source of carbon in the bacterium's natural environment.

This enzyme from Flavobacterium heparinum is highly activated to chondroitin 4-sulfate and chondroitin 6-sulfate. These GAGs are major components of the extracellular matrix. The enzyme consists of two domains, a C-terminal domain containing a 4-beta-sheet sandwich, and an N-terminal domain containing the active site which is made up of alpha-helices. Although calcium is known to be activating, it is not yet clear if it is actively involved in the catalytic mechanism or in substrate binding/recognition.

 

Reference Protein and Structure

Sequence
Q59288 UniProt (4.2.2.5) IPR008929 (Sequence Homologues) (PDB Homologues)
Biological species
Pedobacter heparinus DSM 2366 (Bacteria) Uniprot
PDB
1cb8 - CHONDROITINASE AC LYASE FROM FLAVOBACTERIUM HEPARINUM (1.9 Å) PDBe PDBsum 1cb8
Catalytic CATH Domains
1.50.10.100 CATHdb 2.70.98.10 CATHdb (see all for 1cb8)
Click To Show Structure

Enzyme Reaction (EC:4.2.2.5)

beta-D-GalpNAc4S-(1->4)-beta-D-GlcpA-(1->3)-beta-D-GalpNAc4S
CHEBI:63855ChEBI
→
N-Acetylgalactosamine 4-sulfate
CHEBI:80408ChEBI
+
2-acetamido-2-deoxy-3-O-(4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid)-4-O-sulfo-D-galactopyranose
CHEBI:15931ChEBI
Alternative enzyme names: Chondroitin AC eliminase, Chondroitin sulfate lyase, Chondroitinase, Chondroitinase AC,

Enzyme Mechanism

Introduction

A general base (Tyr234) abstracts the proton from C-5 of the uronic acid, generating an enolate anion intermediate, followed by proton donation by a general acid or water molecule and concomitant beta-elimination of the leaving group. Proton abstraction and beta-elimination are expected to proceed stepwise, as opposed to a concerted manner

Catalytic Residues Roles

UniProt PDB* (1cb8)
Tyr234 Tyr234(212)A Thought to act as a general acid/base catalyst, negatively charged in the enzyme ground state. Abstracts the proton from C-5 of the glucuronic acid at the +1 subsite and donates the abstracted proton back to the leaving group. proton shuttle (general acid/base)
Arg288 Arg288(266)A Contributes to charge neutralization and stabilization of the enolate anion intermediate during catalysis. modifies pKa, electrostatic stabiliser
His225 His225(203)A Has two roles: (1) it helps properly orient the carboxylate group of the glucuronic acid through the NE2---O6A hydrogen bond; (2) being protonated in the complex and in conjunction with Arg296, it lowers the pKa of Tyr242 leading to the deprotonation of its hydroxyl group and priming it for the role as a general base. modifies pKa, electrostatic stabiliser
Glu371 Glu371(349)A Contributes to the stabilisation of the charge state in the active site and the perturbation of the pKa of Tyr234. modifies pKa
Asn175 Asn175(153)A Stabilises the intermediate and transition states. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Rye CS et al. (2002), J Am Chem Soc, 124, 9756-9767. Elucidation of the Mechanism of Polysaccharide Cleavage by Chondroitin AC Lyase fromFlavobacteriumheparinum. DOI:10.1021/ja020627c.
  2. Rye CS et al. (2006), Chembiochem, 7, 631-637. An Atypical Approach Identifies TYR234 as the Key Base Catalyst in Chondroitin AC Lyase. DOI:10.1002/cbic.200500428. PMID:16521140.
  3. Huckerby TN et al. (2005), FEBS J, 272, 6276-6286. Characterization of oligosaccharides from the chondroitin/dermatan sulfates. 1H-NMR and 13C-NMR studies of reduced trisaccharides and hexasaccharides. DOI:10.1111/j.1742-4658.2005.05009.x. PMID:16336265.
  4. Lunin VV et al. (2004), J Mol Biol, 337, 367-386. High-resolution Crystal Structure of Arthrobacter aurescens Chondroitin AC Lyase: An Enzyme–Substrate Complex Defines the Catalytic Mechanism. DOI:10.1016/j.jmb.2003.12.071. PMID:15003453.
  5. Capila I et al. (2002), Biochim Biophys Acta Protein Struct Mol Enzymol, 1597, 260-270. Role of arginine 292 in the catalytic activity of chondroitin AC lyase from Flavobacterium heparinum. DOI:10.1016/s0167-4838(02)00304-7.
  6. Huang W et al. (2001), Biochemistry, 40, 2359-2372. Active Site of Chondroitin AC Lyase Revealed by the Structure of Enzyme−Oligosaccharide Complexes and Mutagenesis†,‡. DOI:10.1021/bi0024254.
  7. Féthière J et al. (1999), J Mol Biol, 288, 635-647. Crystal structure of chondroitin AC lyase, a representative of a family of glycosaminoglycan degrading enzymes. DOI:10.1006/jmbi.1999.2698. PMID:10329169.

Step 1.

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Catalytic Residues Roles

Residue Roles
Tyr234(212)A proton shuttle (general acid/base)
His225(203)A electrostatic stabiliser
Arg288(266)A modifies pKa, electrostatic stabiliser
His225(203)A modifies pKa
Glu371(349)A modifies pKa
Asn175(153)A electrostatic stabiliser

Chemical Components

Step 1.

Contributors

Nozomi Nagano, Gemma L. Holliday, Craig Porter